Additional file 2: of Genomic data mining reveals a rich repertoire of transport proteins in Streptomyces

The classification of Streptomyces transporters. (XLSX 76 kb

Additional file 1: of Genomic data mining reveals a rich repertoire of transport proteins in Streptomyces

A detailed description of transporters in eleven Streptomyces genomes. The file includs protein IDs, names, annotations, protein lengths, Pfam domains, number of TMSs, and their homologs in TCDB with the BLASTP E-value. (XLSX 918 kb

Table_1.doc

<p>AdpA, an AraC/XylS family protein, had been proved as a key regulator for secondary metabolism and morphological differentiation in Streptomyces griseus. Here, we identify AdpA_ch, an ortholog of AdpA, as a “higher level” pleiotropic regulator of natamycin biosynthesis with bidirectional regulatory ability in Streptomyces chattanoogensis L10. DNase I footprinting revealed six AdpA_ch-binding sites in the scnRI–scnRII intergenic region. Further analysis using the xylE reporter gene fused to the scnRI–scnRII intergenic region of mutated binding sites demonstrated that the expression of scnRI and scnRII was under the control of AdpA_ch. AdpA_ch showed a bi-stable regulatory ability where it firstly binds to the Site C and Site D to activate the transcription of the two pathway-specific genes, scnRI and scnRII, and then binds to other sites where it acts as an inhibitor. When Site A and Site F were mutated in vivo, the production of natamycin was increased by 21% and 25%, respectively. These findings indicated an autoregulatory mechanism where AdpA_ch serves as a master switch with bidirectional regulation for natamycin biosynthesis.</p

Proposed schematic model of <i>secDF</i> homologs evolution in <i>Streptomyces</i>.

<p>Proposed schematic model of <i>secDF</i> homologs evolution in <i>Streptomyces</i>.</p

Phylogeny and gene synteny of <i>Streptomyces</i> SecDF homologs.

<p>(A) Phylogenetic tree based on the amino acid sequences of SecDF homologues identified by BLAST searches (see Methods) from those species with whole genome sequences available. Background colors refer to two different clades of SecDF and SecD-F. Bootsrtap support values greater than 50% are depicted in tree branches. Streptomyces branches are shown red, Actinobacteria branches are blue and <i>Dehalobacter</i> branches are pink. Species names are abbreviated as given in Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105237#pone.0105237.s001" target="_blank">File S1</a>. (B) Chromosome regions encompassing secDF gene are depicted by black lines for some species in the phylogenetic tree. The <i>secD</i> or <i>secDF</i> homologous genes are shown as red arrow in the center, and the orthologous genes between these species are depicted in the same color according to IMG database (see Methods).</p

The gene knock out processes and gene expression profiles of <i>secDF</i> homologous genes in <i>S. coelicolor</i>.

<p>(A) Schematic representation of the in frame deletion of <i>secD-F</i> and <i>secDF</i> by double-crossover homologous recombination in <i>S. coelicolor</i> M145. Plasmids pKC1139a and pKC1139b were used to knock out <i>secD-F</i> (ZJUZ1) and <i>secDF</i> (ZJUZ2), respectively. Both pKC1139a and pKC1139b were used to create the ZJUZ3, which lacks both <i>secD-F</i> and <i>secDF</i> genes. (B) Gene expression analysis of <i>secD</i>, <i>secF</i> and <i>secDF</i> in <i>S. coelicolor</i> wild type and mutant strains by RT-PCR with 30 and 35 cycles. Gene hrdB was used as a reference gene.</p

qPCR analysis of gene expression profiles of <i>secD</i>, <i>secF</i> and <i>secDF</i> in <i>S. coelicolor</i> wild type and mutant strains.

<p>(A) qPCR analysis of gene expression profile of <i>secD</i>, <i>secF</i> and <i>secDF</i> in <i>S. coelicolor</i> M145 alone the time of cultivation. (B) qPCR analysis of <i>secD</i> and <i>secF</i> gene expression in M145 and ZJUZ2. (C) qPCR analysis of <i>secDF</i> gene expression in M145 and ZJUZ1. Gene <i>hrdB</i> was used as a reference gene.</p

Strains and plasmids used in this study.

<p><i>ermEp*</i>: the enhanced promoter region of the erythromycin resistance gene (<i>ermE</i>) of <i>Streptomyces erythraeus</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105237#pone.0105237-Bibb1" target="_blank">[48]</a>.</p

Assay of extracellular XlnA and AmlC activity and protein amounts.

<p>(A) Detecting XlnA protein secretion by SDS-PAGE and Western blot. WB: Western blot; CB: Commassie Blue Staining. ZJUZ23-26 strains were cultivated for 96 h. (B) Assay of extracellular XlnA activity. ZJUZ23-26 strains were cultivated for 72 h and 96 h. *: p<0.05, **: p>0.05, Chi-squared test. (C) Relative amylase activity from ZJUZ27, ZJUZ28, ZJUZ29 and ZJUZ30. The diameters of the transparent zones around colonies were measured to determine the relative activity.</p